Nowadays the biotin/streptavidin system is a generally known binding system in molecular biology the importance of which has increased considerably in recent years and which is used in various fields of application. In doing so one utilizes the specific affinity between biotin and streptavidin which, together with an affinity constant of the order of 1013, is one of the most stable known non-covalent interactions.
Important conventional applications are for diverse separation and detection methods usually using biotinylated enzymes or/and antibodies in various variations. Examples are for example ELISA, Western blot etc. A prerequisite for such methods is that the reagent or enzyme used in a biotinylated form in the method must firstly be obtainable in a pure form in order to be able to carry out the biotinylation which takes place in a chemical reaction.
However, for certain applications a biotinylation is not possible or at least not in a simple manner such as for example when detecting and purifying recombinantly produced proteins which have previously not yet been isolated. Therefore in the past methods for modifying the biotin/streptavidin system have been sought in order to extend its range of application.
A successful approach has been to produce peptide ligands which also have a specific binding affinity for streptavidin. Suitable peptide ligands and corresponding fusion proteins are disclosed in DE-OS 42 37 113. The advantage of these peptide ligands compared to biotin is essentially that their coding sequence is linked at the DNA level with the gene of a desired protein and can subsequently be coexpressed together with that of the protein by which means a recombinant protein labelled with the peptide ligand, i.e. fused thereto, is formed. Due to the small size of the peptide ligands and the fact that they can be attached to the N- or C-terminus of the desired protein, i.e. in areas which often are not of major importance for the structure and biochemical function of the protein, it is generally also not necessary to again cleave off the peptide ligand after its isolation and before using the protein for other purposes so that this also results in a more economical process. Indeed no case is yet known in which a cleavage would have been necessary. If nevertheless cleavage should be necessary, this can be accomplished by inserting a protease cleavage site between the binding peptide and protein sequence.
Such peptide ligands which are suitable are described in detail for example in Schmidt and Skerra, Protein Eng. 6 (1993), 109-122 and J. Chromatogr. A 676 (1994), 337-345 as well as in Schmidt et al., J. Mol. Biol. 255 (1996), 753-766.
Advantages of the streptavidin peptide ligand system are that the purification of recombinant proteins becomes possible at all and that this purification can be achieved for example by affinity chromatography under very mild elution conditions since the bound peptide ligand as part of the recombinant protein is displaced competitively by biotin or derivatives thereof. In addition the peptide ligand enables the recombinant protein to be for example detected by Western blot, ELISA or by immune microscopy using suitable streptavidin conjugates.
A disadvantage of this system has previously been its relatively low affinity. An affinity constant of 2.7.times.10.sup.4 M.sup.-1 has been determined by means of isothermal titration calorimetry for the complex between streptavidin and the peptide ligand referred to as strep-tag (Ala Trp Arg His Pro Gln Phe Gly Gly (SEQ ID NO: 1)). Although there were indications that the binding could be somewhat stronger for a fusion protein containing the peptide ligand, it is desirable to have a system with a fundamentally improved affinity.
Hence the object of the invention was to optimize the streptavidin/peptide ligand system with regard to binding strength.
After initial experiments had been carried out to further optimize the sequence of the peptide ligand, it had to be assumed that the peptide ligand according to DE-OS-4237113 already apparently represented an optimum and thus this approach was less promising.
Once the crystal structure of the streptavidin/peptide ligand complex was available in high resolution, a better understanding was gained of the molecular interactions and the structural characteristics (Schmidt et al. (1996), supra) but no clear information could be obtained from these structural data on whether and in which manner a modification of the peptide sequence or of streptavidin could be carried out in a rational manner to improve the affinity and hence to achieve the initial objective.
In an evolutionary research approach it has now been surprisingly found that the binding affinity for the streptavidin/peptide ligand system can be improved by mutation in the region of the amino acid positions 44 to 53 of streptavidin.